A large unmet medical need exists for safe, well-tolerated and effective drugs for the treatment of patients with cystic fibrosis (CF), COPD/emphysema, bronchiectasis, severe asthma and other serious obstructive pulmonary diseases. These diseases are characterized by overproduction of thickened mucus resulting in impaired lung function (reviewed in Evans, C. M. and Koo, J. S., Pharmacology & Therapeutics 121: 332-348, 2009). Poor clearance of abnormal, sticky mucus is associated with chronic infection and premature death, especially in CF. Despite advances in antibiotic therapy and other treatments, improved mucus clearance remains a central clinical treatment objective even while our understanding of the mechanisms underlying mucus transportability are still limited (Verdugo, P., Cold Spring Harb Perspect Med 2012; 2:a009597).
Mucus is a continuously-secreted supramolecular polymer gel that forms a protective barrier on epithelial surfaces and is responsible via ciliary action and cough for transporting inhaled debris and bacteria out of the lung (Knowles, M. R. and Boucher, R. C., J Clin Invest 109:571-577, 2002; Cone, R. A., Adv Drug Deliv Rev 61:75-85, 2009). Proper viscoelasticity and hydration of the mucus layer, which enables efficient mucociliary transport, is therefore critical to mucus function and the prevention of infection and inflammation. Normal mucus consists of mostly water (97%) with the remaining solids comprising mucin proteins, non-mucin proteins, salts, lipids and cellular debris (Fahy, J. V. and Dickey, B. F., N Engl J Med 363:2233-47, 2010). The polymeric mucin glycoproteins MUC5AC and MUC5B are primarily responsible for the viscoelastic properties of the respiratory mucus gel (Matsui et al., Cell 95:1005-1015, 1998; reviewed in Kreda, et al., Cold Spring Harb Perspect Med 2012; 2:a009589). The O-linked glycan hydroxyl groups attached to mucins contribute water-binding, while the mucins themselves form an entangled network (Verdugo et al., Biorheology 20:223-230, 1983) that may also involve covalent and non-covalent interchain cross-linking as suggested from detailed studies of the digestive tract mucin MUC2 (Ambort et al., Biochem J 436:61-70, 2011). Mucins are unusually rich in Cys amino acids, with human MUC5AC containing a remarkable 295 Cys residues out of a total of 5030 amino acids (www.uniprot.org/uniprot/P98088). Mucin Cys residues located near the N and C termini are thought to be involved in formation of interchain disulfide linkages between mucin subunits, while the role of the internal Cys residues is less clear (Thornton et al., Annu Rev Physiol 70:459-486, 2008). Some are located in a ‘Cys Knot’ region and might readily form intramolecular disulfide bonds that could play a role in facilitating the non-covalent entanglement central to the mucus gel mesh structure (Fahy, J. V. and Dickey, B. F., N Engl J Med 363:2233-47, 2010).
Recent work (Button et al., Science 337:937-941, 2012) has led to a new model of the structure of the mucosal surface based on the finding that certain mucins, once thought to be membrane bound on epithelial cells, are actually tethered to membranes on cilia themselves. The implication of this model is that the mobile mucus layer overlays an even denser periciliary layer, described as a “gel-on-brush”. The model explains elegantly how liquid moves between the two layers with the mucus acting as a reservoir, and establishes a new paradigm for understanding the role of mucus osmotic modulus in determining the functionality of mucociliary transport and mucus layer hydration. The model also provides a framework to understand how excess disulfide bonding in the mucus protein scaffold might cause increased mucus layer osmotic modulus, which in turn dehydrates the underlying ciliary layer and severely constrains normal mucus transport. Such a scenario may underpin a substantial portion of the disease mechanism of CF.
CF is an autosomal recessive condition. The symptoms of CF result from defects in CFTR, the Cystic Fibrosis Transmembrane Conductance Regulator, a key epithelial membrane transporter for monovalent negatively charged ions, primarily chloride (Riordan, et al., Science 245: 1066-1073), but also bicarbonate and glutathione. Mutations leading to CF, of which over 1700 are known (www.genet.sickkids.on.ca/cftr/), include those causing complete loss of CFTR (the case for the most common CF genotype) as well as point mutations resulting in partial or full loss of anion transport activity. In addition, as a result of the defects in CFTR, epithelia within the body are impermeable to chloride ion transport (Boucher et al., Lung 161:1-17, 1983; Welsh, Physiol Rev 67:11443-1184, 1987). Although several organs are affected, including pancreas, intestine, and male genital tract, complications within the lung account for 95% of the morbidity and mortality (Means, M. Cystic Fibrosis: the first 50 years. In: Cystic Fibrosis-Current Topics Volume 1, edited by Dodge J A, Brock D J H, and Widdicombe J H. Chichester: Wiley and Sons, 1992, p. 217-250). In lung impaired by the disease, chloride transport into the airway lumen leads to excessive absorption of Na+ and fluid, reducing the volume of airway surface liquid (Jiang et al., Science 262:424-427, 1993). Attempts have failed, however, to restore chloride channel activity to compensate for the effects caused by the non-functioning CFTR, e.g. via agonists of the P2Y2 subtype of purinergic receptor (Ratjen, F. et al., J Cyst Fibros 11:539-49, 2012). This suggests that non-chloride effects of CFTR might be more significant than originally thought.
In oxidizing environments like the lung, disulfide bonds are readily formed between adjacent oxidized Cys residues such as those present in great abundance on mucin proteins. These bonds are highly stable, and disrupting (i.e. reducing) them in order to restore the Cys residues to their free thiol form requires the action of potent chemical or biological reductants. In the healthy lung, excess disulfide bond formation is countered primarily by the reduced form of the biological reductant glutathione (GSH), a Cys-containing tripeptide that is secreted in large amounts into the mucus layer (Cantin et al., J Appl Physiol 63:152-157, 1987), and may play a key role in maintaining a normal disulfide bond vs. free Cys thiol equilibrium in mucins. Secretion of GSH onto the airway surface is highly dependent upon CFTR, which both directly and indirectly facilitates GSH export (reviewed in Ballatori et al., Biol Chem 390: 191-214, 2009). Consequently, levels of pulmonary GSH in CF patients may be 30% or less than levels found in normal individuals (Roum et al., J Appl Physiol 75:2419-24, 2003; Wetmore D. R. et al., J Biol Chem 285:30516-22, 2010). CFTR is also responsible for secretion of bicarbonate anions, and the resulting deficiency of bicarbonate in the CF lung appears to contribute to disease. A primary chemical effect of bicarbonate is to raise pH. Since reduction of disulfide bonds by thiol-containing reductants requires the formation of an attacking deprotonated thiolate, which is inhibited at low pH where the protonated thiol form is favored (Singh and Whitesides, In: Sulphur-containing Functional Groups, 5: pp. 633-58, John Wiley & Sons, 1993), synergy between the activities of bicarbonate and GSH (as well as other biological reductants known to be present in the airway surface environs) is likely. Measured pH in CF tracheobronchial secretions is up to 0.6 units lower vs non-diseased (Song et al., Am J Physiol Cell Physiol 290:C741-C749), consistent with an environment in the CF lung where reductant is both present in limiting supply as well as being less active due to an impaired ability to form disulfide-attacking thiolates. Taken together with the enormous number of clustered Cys present in mucins, mucus in the oxidizing respiratory environment is thus poised to be in a more highly disulfide-bonded state if either secreted reductant levels become limiting, or if mucin proteins are produced and secreted in excess resulting in a superabundance of disulfide-bondable Cys. Both situations are known to occur in CF and certain other obstructive pulmonary diseases: mucus proteins are over-produced in response to lung stress (Rogers, Resp Care 52:1134-1149), and 70% or more of GSH secretion can be blocked as a consequence of defects in CFTR (Roum et al., J Appl Physiol 75:2419-24, 2003; Wetmore D. R. et al., J Biol Chem 285:30516-22, 2010).
This potential for excess mucus disulfide-bonding as well as general redox imbalance to play a mechanistic role in CF has led to the clinical evaluation of various thiol-containing agents as mucolytic drugs. These include N-acetylcysteine (NAC) and Nacystelyn (NAL; N-acetylcysteine+L-lysine) (Hurst et al., Am Rev Respir Dis, 96:962-970, 1967; Dasgupta and King, Pediatr Pulmonol, 22:161-166, 1996; Nash, E. F., et al., Cochrane Database of Systematic Reviews, 2010(12): 1-49, 2009) as well as reduced glutathione itself (Bishop, C., et al., CHEST Journal, 127(1): 308-317, 2005; Griese, M., et al., Am J Resp Crit Care Med 169(7):822-828, 2004; Griese, M., et al., Am J Resp Crit Care Med 188(1):83-89, 2013; Roum, J. H., et al., J Appl Physiol, 87:438-443, 1999). While largely safe, to date these small-molecule agents have not exhibited clear clinical benefits in either oral or inhaled forms (reviewed in Nash, E. F., et al., Cochrane Database of Systematic Reviews, 2010(12): 1-49, 2009). Much of this lack of efficacy may be the result of low potency or loss of activity during delivery because of autoxidation effects, as well as the potential for inactivation by pulmonary enzymes. GSH is subject to rapid autoxidation to the inactive GSSG form (Curello, S. et al., Clin Chem, 33:1448-49, 1987) and is hence pharmacologically unstable in the reduced form when aerosolized and inhaled (Carl White M. D., pers comm.), losing a large fraction of its activity by the time the target site in the airway is reached. In addition, γ-glutamyltransferase present at high concentrations in the pulmonary space readily degrades GSH to an inactive form (Corti et al., Am J Resp Crit Care Med 189:233-234, 2014), the abundance of which increases markedly upon GSH inhalation (Griese et al., Am J Resp Crit Care Med 188:83-89 Supplemental information, 2013). Improving thiol agents by combining disulfide-targeting with the superior pharmacology and specificity of biologic drugs is thus a key unmet therapeutic objective.
While the etiology of CF lung disease can be attributed to the altered rheological properties of mucus, compromised lung function is rarely evident at birth. Instead, bronchiectasis and airway obstruction progress with age of patient. This chronic lung injury results from a persistent cycle of bacterial infection and inflammatory response. Airway damage results when neutrophils recruited into the lung release matrix degrading enzymes, such as elastase, and harmful reactive oxygen species (reviewed in Konstan and Berger, Pediatr Pulmonol 24:137-142, 1997). Following persistent infection, interaction of mucins with DNA (Potter et al., Am J Dis Child 100:493-495, 1960; Lethem et al., Am Rev Respir Dis 100:493-495, 1990; Lethem et al., Eur Respir J 3:19-23, 1990) and f-actin polymers (Sheils et al., Am J Path 148:919-927, 1996; Tomkiewicz et al., DNA and actin filament ultrastructure in cystic fibrosis sputum. In: Cilia, mucus, and mucociliary interactions, edited by Baum G L, Priel Z, Roth Y, Liron N, and Ostfeld E J. New York, N.Y.: Marcel Dekker, 1998) released from dying inflammatory cells may also occur, and can be responsible for some of the dense and viscous nature of CF sputum in severe disease. The inability to clear such mucus by cough or mucociliary clearance facilitates further colonization of the lung with opportunistic pathogens, airway remodeling, and eventually death.
Interventions designed to mitigate directly the consequences of CFTR defects therefore are particularly desirable, as these may prevent or attenuate disease progression. While direct correction of CF by gene therapy is not yet attainable, the use of potentiator and corrector therapies to restore some degree of CFTR function to defective proteins has been demonstrated recently (Sloane, P A and Rowe, S M, Current Opinion in Pulmonary Medicine 16: 591-7, 2010). Such therapy is limited to a small percentage of CF patients with a particular CFTR defect, such as the G551D mutation targeted by ivacaftor/Kalydeco™ (Jones, A M and Helm, J M, Drugs 69: 1903-10, 2009). However, in these few individuals dramatic results have been observed (Accurso, F J; Rowe, S M; Clancy, J P; Boyle, M P; Dunitz, J M; Durie, P R; Sagel, S D; Hornick, D B et al., The New England Journal of Medicine 363: 1991-2003, 2010) demonstrating that mechanistic intervention in CF is capable of mitigating late-stage consequences such as those resulting from chronic infection and inflammation. Currently, however, symptomatic rather than disease-modifying approaches including antibiotic regimens coupled with drugs that facilitate the clearance of purulent airway secretions remain the mainstay treatments for progressive airway disease. Inhalation of purified rhDNase (Pulmozyme™; Genentech, USA), which digests extracellular DNA present in the CF airway, is widely used as a respiratory decongestant. Such treatment is clinically effective for diminishing sputum viscosity and stabilizing the forced expiratory volume (FEV) (Fuchs et al., N Engl J Med 331:637-642, 1994). Other investigative therapies aimed at breaking down mucin or actin polymers, including N-acetylcysteine (NAC), nacystelyn (an N-acetyl-L-cysteine derivative), and gelsolin, can also reduce sputum viscosity experimentally, but have yet to demonstrate clinical efficacy and attain approval for treatment of CF in the United States (Nash, E F et al., Cochrane Database of Systematic Reviews, 2010(1):CD007168, 2009).
Other approaches being utilized to improve mucus clearance include mucoactive agents such as inhaled hypertonic saline and inhaled high-dose mannitol (Fahy, J. V. and Dickey, B. F., N Engl J Med 363:2233-47, 2010). These agents are thought to act by pulling water osmotically into the mucus layer to increase hydration, or to improve clearance through induction of coughing reflexes. Some evidence exists for both mechanisms (Levin, M. H. et al., J Biol Chem 281:25803-12, 2006; Boucher, R. C., Trends Mol Med 13:231-240, 2007). However, mucoactives are symptomatic treatments (not disease-modifying), and efficacy is generally only moderate as many patients are not able to tolerate the high doses that can have the greatest clinical effect (Aziz, I. and Kastelik, J. A., N Engl J Med 354:1848-1851, 2006).
Findings by White and colleagues (Rancourt et al., Am J Physiol Lung Cell Mol Physiol 286:L931-L938, 2004; Rancourt et al., Free Radical Biol & Med 42:1441-43, 2007) have found that the use of a protein or peptide containing a thioredoxin active site in the reduced state is useful for increasing the liquefaction of mucus or sputum in a patient that has excessively viscous or cohesive mucus or sputum, including a patient having CF, wherein the mucus or sputum is contacted with the protein or peptide (U.S. Pat. No. 7,195,766 and U.S. Pat. No. 7,534,438, both of which are incorporated herein by reference in their entirety). In this system (see FIG. 3), a transient mixed-disulfide between the N-terminal cysteine of the thioredoxin active site and a cysteine of a target protein (found in the mucus or sputum) is formed, followed immediately by nucleophilic attack on the intramolecular mixed disulfide linkage and release of oxidized thioredoxin and the fully-reduced target (Wynn et al., Biochemistry 34(37):11807-11813, 1995), thus allowing for re-formation of cysteine disulfides in the mucus or sputum but at the same time also allowing free access of reduced or oxidized thioredoxin to enter cells and induce undesired off-target activities following re-reduction by the endogenous thioredoxin reductase—NADPH system. In addition White and colleagues have shown reduced thioredoxin to mitigate the abnormal viscoelasticity of human CF mucus in vitro and in ex vivo animal implantation studies (Rancourt et al., Free Radical Biol & Med 42:1441-43, 2007), as well as to inhibit the activity of pro-inflammatory neutrophil elastase by disruption of active site disulfide bonds (Lee et al., Am J Physiol Lung Cell Mol Physiol 289:L875-L882, 2005). Compared to GSH and thiol agents such as NAC, thioredoxin is a more potent disulfide bond-reducing molecule and is much less susceptible to inactivation by autoxidation. Taken together, this creates the opportunity to restore a normal disulfide reduction state to mucus with a pharmacologically stable molecule. Such a therapy may prevent or delay the cascade of chronic infection, inflammation and lung function decline that leads to early death in CF patients. However, there is also a strong motivation to avoid the potential pro-inflammatory and other intracellular regulatory effects of thioredoxin (Arner, E. S. and A. Holmgren, Eur J Biochem 267: 6102-6109, 2000; Rancourt et al., Free Radical Biol & Med 42:1441-43, 2007) as well as to increase the potency of mucus viscosity-modulation by preventing mucin Cys re-oxidation. These improvements are the subject of the present invention.